Hidden hotspots of amphibian biodiversity in China.

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

Large-scale snake genome analyses provide insights into vertebrate development.

Snakes are a remarkable squamate lineage with unique morphological adaptations, especially those related to the evolution of vertebrate skeletons, organs, and sensory systems. To clarify the genetic underpinnings of snake phenotypes, we assembled and analyzed 14 de novo genomes from 12 snake families. We also investigated the genetic basis of the morphological characteristics of snakes using functional experiments. We identified genes, regulatory elements, and structural variations that have potentially contributed to the evolution of limb loss, an elongated body plan, asymmetrical lungs, sensory systems, and digestive adaptations in snakes. We identified some of the genes and regulatory elements that might have shaped the evolution of vision, the skeletal system and diet in blind snakes, and thermoreception in infrared-sensitive snakes. Our study provides insights into the evolution and development of snakes and vertebrates.

Developmental regulation of conserved non-coding element evolution provides insights into limb loss in squamates.

Limb loss shows recurrent phenotypic evolution across squamate lineages. Here, based on three de novo-assembled genomes of limbless lizards from different lineages, we showed that divergence of conserved non-coding elements (CNEs) played an important role in limb development. These CNEs were associated with genes required for limb initiation and outgrowth, and with regulatory signals in the early stage of limb development. Importantly, we identified the extensive existence of insertions and deletions (InDels) in the CNEs, with the numbers ranging from 111 to 756. Most of these CNEs with InDels were lineage-specific in the limbless squamates. Nearby genes of these InDel CNEs were important to early limb formation, such as Tbx4, Fgf10, and Gli3. Based on functional experiments, we found that nucleotide mutations and InDels both affected the regulatory function of the CNEs. Our study provides molecular evidence underlying limb loss in squamate reptiles from a developmental perspective and sheds light on the importance of regulatory element InDels in phenotypic evolution.

Genetic mapping and molecular mechanism behind color variation in the Asian vine snake.

BACKGROUND: Reptiles exhibit a wide variety of skin colors, which serve essential roles in survival and reproduction. However, the molecular basis of these conspicuous colors remains unresolved. RESULTS: We investigate color morph-enriched Asian vine snakes (Ahaetulla prasina), to explore the mechanism underpinning color variations. Transmission electron microscopy imaging and metabolomics analysis indicates that chromatophore morphology (mainly iridophores) is the main basis for differences in skin color. Additionally, we assemble a 1.77-Gb high-quality chromosome-anchored genome of the snake. Genome-wide association study and RNA sequencing reveal a conservative amino acid substitution (p.P20S) in SMARCE1, which may be involved in the regulation of chromatophore development initiated from neural crest cells. SMARCE1 knockdown in zebrafish and immunofluorescence verify the interactions among SMARCE1, iridophores, and tfec, which may determine color variations in the Asian vine snake. CONCLUSIONS: This study reveals the genetic associations of color variation in Asian vine snakes, providing insights and important resources for a deeper understanding of the molecular and genetic mechanisms related to reptilian coloration.

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs.

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.

Effects of climate change and anthropogenic activity on ranges of vertebrate species endemic to the Qinghai-Tibet Plateau over 40 years.

Over the past 40 years, the climate has been changing and human disturbance has increased in the vast Qinghai-Tibet Plateau (QTP). These 2 factors are expected to affect the distribution of a large number of endemic vertebrate species. However, quantitative relationships between range shifts and climate change and human disturbance of these species in the QTP have rarely been evaluated. We used occurrence records of 19 terrestrial vertebrate species (birds, mammals, amphibians, and reptiles) occurring in the QTP from 1980 to 2020 to quantify the effects of climate change and anthropogenic impacts on the distribution of these 4 taxonomic groups and estimated species range changes in each species. The trend in distribution changes differed among the taxonomic groups, although, generally, ranges shifted to central QTP. Climate change contributed more to range variation than human disturbance (the sum of the 4 climatic variables contributed more than the sum of the 4 human disturbance variables for all 4 taxonomic groups). Suitable geographic range increased for most mammals, amphibians, and reptiles (+27.6%, +18.4%, and +27.8% on average, respectively), whereas for birds range decreased on average by 0.9%. Quantitative evidence for climate change and human disturbance associations with range changes for endemic vertebrate species in the QTP can provide useful insights into biodiversity conservation under changing environments.

En los últimos 40 años, el clima ha cambiado y las perturbaciones humanas han aumentado en la vasta meseta Qinghai-Tíbet (MQT). Se espera que estos dos factores afecten la distribución de un gran número de especies de vertebrados endémicos. Sin embargo, las relaciones cuantitativas entre los cambios del área de distribución y el cambio climático y las perturbaciones humanas en estas especies de la MQT han sido poco evaluadas. Utilizamos los registros de presencia de 19 especies de vertebrados terrestres (aves, mamíferos, anfibios y reptiles) de la MQT tomados entre 1980 y 2020 para cuantificar los efectos del cambio climático y los impactos antropogénicos sobre la distribución de estos cuatro grupos taxonómicos y estimar los cambios en el área de distribución de cada especie. La tendencia en los cambios de distribución difirió entre los grupos taxonómicos, aunque, en general, las áreas de distribución se desplazaron hacia el centro de la MQT. El cambio climático contribuyó más a la variación del área de distribución que las perturbaciones humanas (la suma de las cuatro variables climáticas contribuyó más que la suma de las cuatro variables de perturbaciones humanas para los cuatro grupos taxonómicos). El área de distribución geográfica adecuada aumentó para la mayoría de los mamíferos, anfibios y reptiles (+27.6%, +18.4% y +27.8% en promedio, respectivamente), mientras que para las aves disminuyó en promedio un 0.9%. Las pruebas cuantitativas de la asociación del cambio climático y las perturbaciones humanas con los cambios en el área de distribución de las especies vertebradas endémicas de la MQT pueden aportar información útil para la conservación de la biodiversidad en entornos cambiantes.

Genomic evidence reveals intraspecific divergence of the hot-spring snake (Thermophis baileyi), an endangered reptile endemic to the Qinghai-Tibet plateau.

Understanding how and why species evolve requires knowledge on intraspecific divergence. In this study, we examined intraspecific divergence in the endangered hot-spring snake (Thermophis baileyi), an endemic species on the Qinghai-Tibet Plateau (QTP). Whole-genome resequencing of 58 sampled individuals from 15 populations was performed to identify the drivers of intraspecific divergence and explore the potential roles of genes under selection. Our analyses resolved three groups, with major intergroup admixture occurring in regions of group contact. Divergence probably occurred during the Pleistocene as a result of glacial climatic oscillations, Yadong-Gulu rift, and geothermal fields differentiation, while complex gene flow between group pairs reflected a unique intraspecific divergence pattern on the QTP. Intergroup fixed loci involved selected genes functionally related to divergence and local adaptation, especially adaptation to hot spring microenvironments in different geothermal fields. Analysis of structural variants, genetic diversity, inbreeding, and genetic load indicated that the hot-spring snake population has declined to a low level with decreased diversity, which is important for the conservation management of this endangered species. Our study demonstrated that the integration of demographic history, gene flow, genomic divergence genes, and other information is necessary to distinguish the evolutionary processes involved in speciation.

Genome of Laudakia sacra Provides New Insights into High-Altitude Adaptation of Ectotherms.

Anan's rock agama (Laudakia sacra) is a lizard species endemic to the harsh high-altitude environment of the Qinghai-Tibet Plateau, a region characterized by low oxygen tension and high ultraviolet (UV) radiation. To better understand the genetic mechanisms underlying highland adaptation of ectotherms, we assembled a 1.80-Gb L. sacra genome, which contained 284 contigs with an N50 of 20.19 Mb and a BUSCO score of 93.54%. Comparative genomic analysis indicated that mutations in certain genes, including HIF1A, TIE2, and NFAT family members and genes in the respiratory chain, may be common adaptations to hypoxia among high-altitude animals. Compared with lowland reptiles, MLIP showed a convergent mutation in L. sacra and the Tibetan hot-spring snake (Thermophis baileyi), which may affect their hypoxia adaptation. In L. sacra, several genes related to cardiovascular remodeling, erythropoiesis, oxidative phosphorylation, and DNA repair may also be tailored for adaptation to UV radiation and hypoxia. Of note, ERCC6 and MSH2, two genes associated with adaptation to UV radiation in T. baileyi, exhibited L. sacra-specific mutations that may affect peptide function. Thus, this study provides new insights into the potential mechanisms underpinning high-altitude adaptation in ectotherms and reveals certain genetic generalities for animals' survival on the plateau.

Temperature acclimation in hot-spring snakes and the convergence of cold response.

Animals have evolved sophisticated temperature-sensing systems and mechanisms to detect and respond to ambient temperature changes. As a relict species endemic to the Qinghai-Tibet Plateau, hot-spring snake (Thermophis baileyi) survived the dramatic changes in climate that occurred during plateau uplift and ice ages, providing an excellent opportunity to explore the evolution of temperature sensation in ectotherms. Based on distributional information and behavioral experiments, we found that T. baileyi prefer hot-spring habitats and respond more quickly to warmth than other two snakes, suggesting that T. baileyi may evolve an efficient thermal-sensing system. Using high-quality chromosome-level assembly and comparative genomic analysis, we identified cold acclimation genes experiencing convergent acceleration in high-altitude lineages. We also discovered significant evolutionary changes in thermosensation- and thermoregulation-related genes, including the transient receptor potential (TRP) channels. Among these genes, TRPA1 exhibited three species-specific amino acid replacements, which differed from those found in infrared imaging snakes, implying different temperature-sensing molecular strategies. Based on laser-heating experiments, the T. baileyi-specific mutations in TRPA1 resulted in an increase in heat-induced opening probability and thermal sensitivity of the ion channels under the same degree of temperature stimulation, which may help the organism respond to temperature changes more quickly. These results provide insight into the genetic mechanisms underpinning the evolution of temperature-sensing strategies in ectotherms as well as genetic evidence of temperature acclimation in this group.

The structural and functional divergence of a neglected three-finger toxin subfamily in lethal elapids.

Bungarus multicinctus is a widely distributed and medically important elapid snake that produces lethal neurotoxic venom. To study and enhance existing antivenom, we explore the complete repertoire of its toxin genes based on de novo chromosome-level assembly and multi-tissue transcriptome data. Comparative genomic analyses suggest that the three-finger toxin family (3FTX) may evolve through the neofunctionalization of flanking LY6E. A long-neglected 3FTX subfamily (i.e., MKA-3FTX) is also investigated. Only one MKA-3FTX gene, which evolves a different protein conformation, is under positive selection and actively transcribed in the venom gland, functioning as a major toxin effector together with MKT-3FTX subfamily homologs. Furthermore, this lethal snake may acquire self-resistance to its ß-bungarotoxin via amino acid replacements on fast-evolving KCNA2. This study provides valuable resources for further evolutionary and structure-function studies of snake toxins, which are fundamental for the development of effective antivenoms and drug candidates.

East palearctic treefrog past and present habitat suitability using ecological niche models.

Ecological niche modeling is a tool used to determine current potential species' distribution or habitat suitability models which can then be used to project suitable areas in time. Projections of suitability into past climates can identify locations of climate refugia, or areas with high climatic stability likely to contain the highest levels of genetic diversity and stable populations when climatic conditions are less suitable in other parts of the range. Modeling habitat suitability for closely related species in recent past can also reveal potential periods and regions of contact and possible admixture. In the east palearctic, there are five Dryophytes (Hylid treefrog) clades belonging to two groups: Dryophytes japonicus group: Clades A and B; and Dryophytes immaculatus group: Dryophytes immaculatus, Dryophytes flaviventris, and Dryophytes suweonensis. We used maximum entropy modeling to determine the suitable ranges of these five clades during the present and projected to the Last Glacial Maximum (LGM) and Last Interglacial (LIG) periods. We also calculated climatic stability for each clade to identify possible areas of climate refugia. Our models indicated suitable range expansion during the LGM for four clades with the exclusion of D. immaculatus. High climatic stability in our models corresponded to areas with the highest numbers of recorded occurrences in the present. The models produced here can additionally serve as baselines for models of suitability under climate change scenarios and indicate species ecological requirements.

Genomic adaptations for arboreal locomotion in Asian flying treefrogs.

SignificanceTo adapt to arboreal lifestyles, treefrogs have evolved a suite of complex traits that support vertical movement and gliding, thus presenting a unique case for studying the genetic basis for traits causally linked to vertical niche expansion. Here, based on two de novo-assembled Asian treefrog genomes, we determined that genes involved in limb development and keratin cytoskeleton likely played a role in the evolution of their climbing systems. Behavioral and morphological evaluation and time-ordered gene coexpression network analysis revealed the developmental patterns and regulatory pathways of the webbed feet used for gliding in Rhacophorus kio.

Transcriptome Analysis Reveals Olfactory System Expression Characteristics of Aquatic Snakes.

Animal olfactory systems evolved with changes in habitat to detect odor cues from the environment. The aquatic environment, as a unique habitat, poses a formidable challenge for olfactory perception in animals, since the higher density and viscosity of water. The olfactory system in snakes is highly specialized, thus providing the opportunity to explore the adaptive evolution of such systems to unique habitats. To date, however, few studies have explored the changes in gene expression features in the olfactory systems of aquatic snakes. In this study, we carried out RNA sequencing of 26 olfactory tissue samples (vomeronasal organ and olfactory bulb) from two aquatic and two non-aquatic snake species to explore gene expression changes under the aquatic environment. Weighted gene co-expression network analysis showed significant differences in gene expression profiles between aquatic and non-aquatic habitats. The main olfactory systems of the aquatic and non-aquatic snakes were regulated by different genes. Among these genes, RELN may contribute to exploring gene expression changes under the aquatic environment by regulating the formation of inhibitory neurons in the granular cell layer and increasing the separation of neuronal patterns to correctly identify complex chemical information. The high expression of TRPC2 and V2R family genes in the accessory olfactory systems of aquatic snakes should enhance their ability to bind water-soluble odor molecules, and thus obtain more information in hydrophytic habitats. This work provides an important foundation for exploring the olfactory adaptation of snakes in special habitats.

MEANGS: an efficient seed-free tool for de novo assembling animal mitochondrial genome using whole genome NGS data.

Advances in next-generation sequencing (NGS) technologies have led to an exponential increase in the number of whole genome sequences (WGS) in databases. This wealth of WGS data has greatly facilitated the recovery of full mitochondrial genomes (mitogenomes), which are vital for phylogenetic, evolutionary and ecological studies. Unfortunately, most existing software cannot easily assemble mitogenome reference sequences conveniently or efficiently. Therefore, we developed a seed-free de novo assembly tool, MEANGS, which applies the trie-search method to extend contigs from self-discovery seeds and assemble a mitogenome from animal WGS data. We then used data from 16 species with different qualities to compare the performance of MEANGS with three other available programs. MEANGS exhibited the best overall performance since it was the only one that completed all tests, and it assembled full or partial mitogenomes for all of the tested samples while the others failed. Furthermore, MEANGS selects superior assembly sequences and annotates protein-coding genes. Thus, MEANGS can be one of the most efficient software for generating high-quality mitogenomes so far, the further use of it will benefit the study on mitogenome based on whole genome NGS data. MEANGS is available at https://github.com/YanCCscu/meangs.

Taxonomic revision of Amolops chunganensis (Pope, 1929) (Amphibia: Anura) and description of a new species from southwestern China, with discussion on Amolops monticola group and assignment of species groups of the genus Amolops.

Amolops chunganensis is a species complex and reported widely from eastern, southern, and southwestern China. Based on molecular data of 19 populations of A. chunganensis sensu lato from China, including the population from Mt. Wuyi (type locality), we recognize A. chunganensis sensu stricto and provide an expanded description based on the topotypic specimens. Combining morphological and molecular data, we describe a new species, Amolops chaochin sp. nov., from southwestern China, which was previously identified as A. chunganensis. The new species is distinguished from all other species in the A. monticola group by: (1) moderate body size, SVL 35.3-39.2 mm in males (n=7), and 50.5-54.4 mm in females (n=7); (2) distinct tympanum, larger than half of eye diameter; (3) small tooth-like projection on anteromedial edge of mandible; (4) circummarginal groove on all fingers; (5) white tubercles on dorsal side of posterior body in both sexes; (6) distinct tubercles on dorsal thigh and white spinose tubercles on dorsal tibia in both sexes; (7) white tubercles on posterior region of tympanum in males; (8) toe webbing reaching disk by dermal fringe on inner side of toe II; (9) vomerine teeth present; (10) transverse bands on dorsal limbs; (11) external vocal sacs present in males. We further reviewed the assignment of Amolops groups, with an overall revision of membership and diagnosis of all species groups.

Geographical range evolution of the genus Polypedates (Anura: Rhacophoridae) from the Oligocene to present.

Currently, the genus Polypedates comprises 26 species distributed in South, Southeast, and East Asia. Because of their relatively low dispersal capability and intolerance to seawater, this genus is ideal for the study of terrestrial range evolution that extends into the island archipelagos of southeastern Asia. In this study, based on data compiled for Polypedatesfrom previous studies and partial mitochondrial and nuclear genes collected in this study, we performed systematic biogeographical analysis. We confirmed a Sundaland origin for the extant genus and showed northward dispersal into mainland Southeast Asia and Asia, which coincided with the timing of paleoclimatic change from the Oligocene to Middle Miocene. Climate fluctuations had a profound impact on species diversification within the genus Polypedates. Furthermore, the Red River did not mediate species exchange between Southeast Asia and mainland Asia until the end of the Miocene, with the sudden onset of northward dispersal in several clades independently at that time. Alternatively, the lineage of widespread insular P. leucomystaxstrongly supports the hypothesis of terrestrial connection between island archipelagos of Southeast Asia during the Mid-Pleistocene paleoclimate fluctuations. Our biogeographical analysis also supports the recent introduction of P. leucomystax to the Philippines and Ryukyus, as previously suggested.

Yellow sea mediated segregation between North East Asian Dryophytes species.

While comparatively few amphibian species have been described on the North East Asian mainland in the last decades, several species have been the subject of taxonomical debates in relation to the Yellow sea. Here, we sampled Dryophytes sp. treefrogs from the Republic of Korea, the Democratic People's Republic of Korea and the People's Republic of China to clarify the status of this clade around the Yellow sea and determine the impact of sea level change on treefrogs' phylogenetic relationships. Based on genetics, call properties, adult morphology, tadpole morphology and niche modelling, we determined the segregated status species of D. suweonensis and D. immaculatus. We then proceeded to describe a new treefrog species, D. flaviventris sp. nov., from the central lowlands of the Republic of Korea. The new species is geographically segregated from D. suweonensis by the Chilgap mountain range and known to occur only in the area of Buyeo, Nonsan and Iksan in the Republic of Korea. While the Yellow sea is the principal element to the current isolation of the three clades, the paleorivers of the Yellow sea basin are likely to have been the major factor for the divergences within this clade. We recommend conducting rapid conservation assessments as these species are present on very narrow and declining ranges.